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Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells
Published on: May 31, 2011
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Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells
Lucy Pigati1, Richard S Kang, Heike Fölsch
1Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
Methods in Cell Biology
|December 4, 2013
Summary
This study details a viral infection protocol for manipulating gene expression in difficult-to-transfect epithelial cells. It enables stable gene knockdown using retroviruses or lentiviruses and subsequent gene expression using adenoviruses.
Area of Science:
- Cell Biology
- Molecular Biology
- Virology
Background:
- Epithelial cells, particularly polarized ones, are challenging to manipulate using transient transfection.
- Gene expression and knockdown are crucial for studying cellular functions.
Purpose of the Study:
- To provide a detailed protocol for using defective viruses to manipulate gene expression in epithelial cells.
- To establish a method for stable gene knockdown and subsequent gene expression analysis in these cells.
Main Methods:
- Utilizing replication-defective adenoviruses for gene expression.
- Employing replication-defective retroviruses or lentiviruses for stable shRNA-mediated gene knockdown.
- Describing infection procedures for both virus types and cell selection.
Main Results:
- The protocol facilitates efficient gene manipulation in hard-to-transfect epithelial cell lines.
- Stable gene knockdown using shRNA (optionally encoding GFP) is achieved.
- Subsequent infection with adenoviruses allows for analysis of gene expression, such as receptor delivery.
Conclusions:
- Defective viral vectors offer a convenient and efficient approach for gene expression and knockdown in epithelial cell research.
- The described protocol serves as a valuable tool for laboratories studying polarized epithelial cells.
